Role of detrusor PDGFRα+ cells in mouse model of cyclophosphamide-induced detrusor overactivity

Cyclophosphamide (CYP)-induced cystitis is a rodent model that shares many features common to the cystitis occurring in patients, including detrusor overactivity (DO). Platelet-derived growth factor receptor alpha positive (PDGFRα+) cells have been proposed to regulate muscle excitability in murine bladders during filling. PDGFRα+ cells express small conductance Ca2+-activated K+ channels (predominantly SK3) that provide stabilization of membrane potential during filling. We hypothesized that down-regulation of the regulatory functions of PDGFRα+ cells and/or loss of PDGFRα+ cells generates the DO in CYP-treated mice. After CYP treatment, transcripts of Pdgfrα and Kcnn3 and PDGFRα and SK3 protein were reduced in detrusor muscle extracts. The distribution of PDGFRα+ cells was also reduced. Inflammatory markers were increased in CYP-treated detrusor muscles. An SK channel agonist, CyPPA, increased outward current and hyperpolarization in PDGFRα+ cells. This response was significantly depressed in PDGFRα+ cells from CYP-treated bladders. Contractile experiments and ex vivo cystometry showed increased spontaneous contractions and transient contractions, respectively in CYP-treated bladders with a reduction of apamin sensitivity, that could be attributable to the reduction in the SK conductance expressed by PDGFRα+ cells. In summary, PDGFRα+ cells were reduced and the SK3 conductance was downregulated in CYP-treated bladders. These changes are consistent with the development of DO after CYP treatment.

www.nature.com/scientificreports/ These cells are located on the edges of smooth muscle bundles and have multiple branches that may couple to and form an electrical syncytium with smooth muscle cells (SMCs) 25,27,28 . The function of PDGFRα + cells in detrusor muscles has been investigated with molecular and electrophysiological techniques. PDGFRα + cells displayed higher current density of small conductance Ca 2+ -activated K + (SK) channels 26,29 , as compared to detrusor SMCs. Activation of SK channels in PDGFRα + cells could provide a stabilizing influence on smooth muscle excitatiiby. We hypothesized that loss-of-function in PDGFRα + cells or SK channels could lead to DO in cystitis. This study describes investigation into the molecular and functional changes that occur in detrusor PDGFRα + cells and how these changes relate to increased detrusor overactivity in CYP-injected bladder.

Downregulation of PDGFRα and SK channel in murine bladders from CYP-induced cystitis.
We compared the transcriptional expression of Pdgfra and Kcnn1-4 in detrusor muscles from CYP-treated and saline-treated mice. Pdgfra and Kcnn3 (SK3) were significantly downregulated (P < 0.01 by unpaired t-test), but neither SK1-2 (Kcnn1 and Kcnn2) nor IK (Kcnn4) were changed significantly after CYP-treatment (n = 4, Fig. 1A,B). Detrusor muscles from CYP-treated mice displayed a significant increase in Il6 and Tnf (inflammatory markers) (n = 4, Fig. 1C) suggesting the onset of bladder inflammation after CYP treatment. We further examined the transcriptional expression of Pdgfra and Kcnn3 in sorted PDGFRα + cells from saline-and CYPtreated PDGFRα/eGFP mice (see "Methods" section). Pdgfra and Kcnn3 were significantly decreased in CYPtreated mice in comparison to mice treated with saline (n = 4, P < 0.01 in both genes, Fig. 1D). Transcriptional changes were also evaluated in detrusor smooth muscle cells (SMC). SMCs were isolated from saline-and CYPtreated smMHC/Cre/eGFP (see "Methods" section) mice, as previously described 26 . Main excitability-related genes in detrusor SMC, Kcnma1 (BK channels) and Cacna1c (L-type Ca 2+ channels) were unchanged in CYPtreated SMC (n = 4, Fig. 1E). Levels of transcripts do not necessarily translate linearly into protein expression. Therefore, we also employed immunohistochemistry and Wes analysis to confirm parallel changes in protein expression. Immunohistochemistry revealed PDGFRα immune-positive cells were down-regulated in CYP-treated detrusor compared with age-matched saline-injected controls ( Fig. 2A,B). Figure 2C shows a negative control image in which the primary antibody was omitted and the muscles were treated with only secondary antibody. Wes analysis showed in a more quantitative way that PDGFRα and SK3 were significantly downregulated in CYP-treated detrusor muscles, as compared to controls (n = 4, Fig. 3). These data are consistent with transcriptional data, and suggested the possibility that down-regulation of Pdgfrα and/or Kcnn3 transcripts could be involved in generation of DO.   Fig. 4A). In the same cells, CyPPA activated outward current at a holding potential of − 40 mV in voltage-clamp (V-C) mode (Fig. 4A, blue dotted line). CyPPA-activated current amplitude averaged 39.3 ± 7.1 pA. Figure 4B shows currents evoked by ramp depolari-  www.nature.com/scientificreports/ zation before (Fig. 4Ba, black trace) and in the presence of CyPPA (Fig. 4Bb, red trace). The resting membrane potentials (RMP) of CYP-treated detrusor PDGFRα + cells were depolarized to − 16 ± 1.4 mV (n = 5, P < 0.01, as compared to untreated control PDGFRα + cells). CyPPA induced less hyperpolarization in CYP-treated detrusor PDGFRα + cells (∆mV 9.1 ± 2.0), as compared to untreated control PDGFRα + cells (n = 5, P < 0.01, Fig. 4C) under current-clamp mode (I = 0) and generated smaller outward currents under V-C mode (5.2 ± 0.9 pA; n = 5, P < 0.01, Fig. 4D) at − 40 mV. These data suggest that SK current density was significantly decreased in PDGFRα + cells isolated from CYP-treated bladders, as compared with current density in control PDGFRα + cells isolated from non-treated bladders.
The effect of SK channel blocker on detrusor muscle contractions in CYP-treated bladders. We examined the effect of an SK channel blocker to compare changes in functional expression of SK channels between saline-treated (control) and CYP-treated bladders using isometric force measurements. Detrusor muscle strips without submucosa exhibited spontaneous contractions. In saline-injected control, apamin (300 nM, a selective blocker of SK channels) dramatically increased AUC from 62.9 ± 7.9 to 261.5 ± 22.5 mN s during 5 min recordings (n = 8, P < 0.0001, Fig. 5A,C). These data are consistent with previous reports [30][31][32] . In CYP-treated detrusor muscle strips, spontaneous contractions were of high amplitude and irregular. AUC for these contractions was calculated since averaging the frequency and amplitude of these irregular contractions are not reliable measurements. AUC in CYP-treated muscles before and after apamin were 315.7 ± 69.8 mN s and 341.3 ± 66.4 mN s (n = 6, Fig. 5B,D), respectively. Thus, apamin had no significant effect on spontaneous contractile activity in CYP-treated muscles. We also calculated the apamin-sensitive contractions by normalized the effect of apamin from control AUC (before apamin). The sensitivity to apamin was significantly decreased in CYP-treated detrusor muscles (1.1 ± 0.1-fold) compared to saline-injected detrusor muscle strips (4.6 ± 0.6 fold, P < 0.001, Fig. 5D).
The effect of SK channel blocker and agonist on CYP-treated bladders using ex vivo preparation. We also examined the pressure-volume relationships of excised bladders in ex vivo preparations to investigate changes in functional expression of SK channel in control and CYP-treated bladders. Ex vivo pressure-volume measurements exclude extrinsic neural regulation during filling, so this technique highlights regulation via myogenic mechanisms. In in vivo cystometry, voiding contractions start around 20 cmH 2 O in murine bladder 33 . Thus, we analyzed the pressure amplitude and frequency up to 15 cmH 2 O (see Fig. 6A

Discussion
This study investigated the mechanisms of DO in CYP-induced cystitis. Gene transcripts and protein levels of PDGFRα and SK3 were depressed in CYP-treated detrusor muscles and in PDGFRα + cells sorted to purity by FACS. The current density from SK channels evoked by a SK channel agonist, CyPPA, was also depressed in PDGFRα + cells isolated from CYP-treated detrusor muscles, suggesting that gene and protein expression levels have functional consequences on bladder function. Contractile experiments supported the idea that apaminsensitive contractions were decreased in CYP-treated detrusor muscle strips. In ex vivo experiments, an SK channel antagonist, apamin, increased the amplitude and frequency of TCs during filling in control bladders. However, the effects of apamin were reduced significantly in CYP-treated bladders. Similarly the effects of an SK agonist were also minimal in CYP-treated bladders. These data suggest that loss of PDGFRα + cells and the major functional conductance provided by SK channels in these cells results in increased TCs and induction of DO in CYP-treated bladders. Chemical cystitis is one of the adverse effects observed after administration of CYP chemotherapy in humans 34 . CYP also induces cystitis in mice and rats 13,35 . Therefore, CYP treatments of rodents has been used widely as an experimental model of IC/BPS. Single CYP intraperitoneal injection leads to urinary bladder inflammation 36 , visceral pain 8 and DO 23 . However, the mechanisms of DO induced by CYP have not been and after apamin in saline-injected (n = 8) and CYP-treated (n = 6) detrusor muscles. (D) Normalized apamin sensitivity from (C) in saline and CYP treated detrusor muscles. ****Denotes P < 0.0001 by paired t-test in (C) and ***denotes P < 0.001 by unpaired t-test in (D). www.nature.com/scientificreports/ clarified by previous studies. In the current study we tested the hypothesis that functional causes of DO after CYP treatment could be related to loss-of-function of bladder regulation provided by PDGFRα + cells.
Previous studies have shown that as the bladder fills, volume increases and the walls are stretched, but intravesical pressure remains low during much of the period of filling 37 . This accommodation occurs even though there is a natural tendency for detrusor SMCs to contract in response to stretch 38,39 . During bladder filling, NVCs are detected in cystometric records from various species [40][41][42][43] . NVCs appear to correspond to localized contractions that are also observed in ex vivo bladder preparations and have been termed 'spontaneous phasic contractions' , 'micromotions' or 'TCs' [44][45][46][47][48] . TCs increase as bladder filling proceeds 44 . Activation of non-selective cation channels expressed in detrusor SMCs 38,39 generates TCs which initiate sensory inputs via activation of afferent nerves 44 that propagate to the central nervous system during bladder filling [49][50][51][52] . However, under physiological condition, development of TCs can be restrained due to activation of SK channels in detrusor muscles 26 . For instance, SK channel activators reduce and SK channel blocker increase detrusor muscle contractions 30,32,[53][54][55] . Furthermore, Kcnn3 knockout mice show an increase in NVCs in in vivo cystometry and TCs in ex vivo bladders 44,56 . Previous reports demonstrate that Kcnn3 are highly expressed in detrusor PDGFRα + cells 26,29,57 . The current density of SK channels is very low in SMCs compared to detrusor PDGFRα + cells 25. Although there is no direct evidence of electrical coupling between PDGFRα + cells and SMCs, it seems clear that PDGFRα + cells may serve an important stabilizing role in regulating contractile activity of SMCs during www.nature.com/scientificreports/ bladder filling. Therefore, disruption of PDGFRα + cells and/or downregulation of SK channels and intracellular signaling pathways regulating SK channel activity could lead to DO.
In the present study we found that Kcnn3 and Pdgfra transcripts were depressed in CYP-treated detrusor muscles and in detrusor PDGFRα + cells from CYP-treated PDGFRα/eGP mice. In contrast, Kcnma1 and Cac-na1C that encode important proteins that have been implicated in the regulation of bladder excitability were unchanged from control SMCs in mice treated with CYP. Western analysis confirmed that both PDGFRα and SK3 proteins were reduced in CYP-treated detrusor muscles.
Immunohistochemistry also showed a reduction in the density of PDGFRα + cells in CYP-injected detrusor muscle. At present we do not know what factors, activated by CYP, might be responsible for loss of PDGFRα + cells and downregulation of SK channels in detrusor muscles, however the upregulation of inflammatory factors, such as Tnfα and Il6, suggest that an inflammatory mechanism may be involved.
Functional studies to evaluate the state of the SK conductance in detrusor PDGFRα + cells before and after CYP treatment were performed using the patch clamp technique. Our recordings confirmed the presence of an SK conductance in control (untreated) PDGFRα + cells, and the availability of this conductance was decreased in PDGFRα + cells isolated from CYP-treated bladders. As above, CYP treatment led to reduced PDGFRα + cell density, and the patch clamp experiments showed that there was concomitant reduction in the SK conductance normally prominent in these cells. Since SK channels provide stabilization of membrane potential and excitability of SMCs during bladder filling, reducing the availability of the SK conductance would lead to increased generation of TCs. This hypothesis was confirmed using isometric force measurments of detrusor muscle strips and ex vivo bladder preparations of mice treated with CYP. We found an increase in TCs as the bladders were filled and less sensitivity of apamin in comparison to the control mice.
In conclusion, we found that CYP treatments induced DO was caused by reduced detrusor PDGFRα + cells and reduction in the prominent SK conductance expressed by these cells that is utilized to regulate SMC excitability during bladder filling. These experiments provide a novel understanding of detrusor PDGFRα + cells and how defects in these cells can contribute to the development of abnormal bladder activity.

Methods
Preparation of tissue. Male C57BL/6J, Pdgfra tm11(EGFP)Sor /J (PDGFRα/eGFP) and smMHC/Cre/eGFP were purchased from Jackson Laboratory, Bar Harbor, ME. The mice were maintained and experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All methods are reported in accordance with ARRIVE guidelines. Animal protocols were approved by the University of Nevada, Reno Institutional Animal Care and Use Committee. Mice were housed in a pathogen-free barrier facility on a 12-h light/dark cycle with free access to water and food (Prolab 5P76 Isopro 3000; 5.4% fat by weight). All mice were males and used at 8-10 weeks of age for all experiments (purchased from Jackson Laboratory, Bar Harbor, ME, USA). Mice were sacrificed with isoflurane inhalation (AErrane; Baxter, Deerfield,

Induction of CYP-induced cystitis.
Murine CYP-induced cystitis was established according to previously described protocols 23,36,58 . To induce acute cystitis, C57BL/6J, Pdgfra tm11(EGFP)Sor /J (PDGFRα/eGFP) and smMHC/Cre/eGFP were injected with 1.2 mg of CYP per 100 μl of saline solution and control mice were injected only with saline solution. Both mice were sacrificed on day 7 after CYP treatment.

RNA isolation, reverse-transcription PCR and quantitative PCR.
For quantitative analysis of transcripts, PDGFRα + cells and SMCs purified by fluorescence-activated cell sorting (FACS), and detrusor muscles were used for molecular tests as previously described 26 . Total RNA was isolated from the detrusor smooth muscle tissues and sorted cells using Direct-zol RNA miniPrep Kit (Zymo Research, Irvine, CA, USA), and firststrand cDNA was synthesized using qScriptTM cDNA SuperMix (Quanta, Gaithersburg, MD, USA) according to the manufacturer's instructions. Endpoint PCR was performed with specific primers (Table 1)  Wes Simple Western automated capillary electrophoresis and immunodetection. Tissue samples were prepared by homogenizing at 4 °C in 0.3 ml radioimmune precipitation assay (RIPA) buffer (1×/type) Table 1. Primer sequences used for qPCR.

Gene name Primer sequences Accession number
Gapdh F-GCC GAT GCC CCC ATG TTT GTGA  R-GGG TGG CAG TGA TGG CAT GGAC  NM_008084.3   Pdgfra  F-ATG ACA GGA GGG AGG GCT TCA ACG  R-CGG CAC AGG TCA CCA CGA TCG TTT  NM_011058.2   Tnf  F-CTG AAC TTC GGG GTG ATC GG  R-GGC TTG TCA CTC GAA TTT TGAGA  NM_013693.3   Il6  F-TCC AGT TGC CTT CTT GGG  www.nature.com/scientificreports/ with added protease inhibitor tablet (Thermo-Fisher mini tablets EDTA free) with a Bullet Blender (5 min, speed 5, 1 stainless steel bead per detrusor muscle). The homogenate was centrifuged at 4 °C, 3000×g for 10 min, to remove cell debris. The supernatant was aliquoted and stored at − 80 °C 59 . Other tissue samples were subjected to differential centrifugation to obtain a plasma membrane-enriched fraction for PDGFRα and SK3 detection. In this case, the tissues were homogenized in ice cold lysis buffer (mM; 50 Tris HCl pH 8.0, 60 beta-glycerophosphate, 100 NaF, 2 EGTA, 25 Na-pyrophosphate, 1 DTT, and protease inhibitor tablet). Each tissue was homogenized in 0.3 ml lysis buffer, centrifuged at 16,000×g at 4 °C for 10 min, and the supernatants centrifuged at 100,000×g for 1 h at 4 °C. The 100,000×g pellet was resuspended into 0.3 ml of lysis buffer, and stored at − 80 °C. Protein concentrations of the supernatants were determined by the Bradford assay using bovine γ-globulin as the standard. Automated western blotting (Wes Simple Western, ProteinSimple, Santa Clara, CA) was utilized to measure PDGFRα and SK3 protein levels. Simple Western analysis was performed according to the Pro-teinSimple user manual. The primary antibodies and total protein lysate concentrations for each protein were determined by initial titrations of lysate amounts and antibody dilutions. Final concentrations of bladder lysates were 1.0 mg/ml and 0.3 mg/ml for SK3 and PDGFRα respectively. The antibody for SK3 (catalog #sc-28621, Santa Cruz Biotechnologies, CA, USA) was used at a1:100 dilution. The antibody for PDGFRα (catalog #sc-338, Santa Cruz Biotechnologies, CA, USA) was used at a 1:100 dilution. The boiled samples, biotinylated protein ladder, blocking buffer, primary antibodies, secondary antibodies, chemiluminescent substrate, and wash buffer were loaded into the plate (Wes 12-230 kDa Pre-filled Plates with Split Buffer, ProteinSimple). The plate was then loaded onto the automatic size-based Simple Western system for protein separation, antibody incubation and imaging using the Wes default parameters. Image reconstruction of the detected proteins was generated by Compass software (ProteinSimple). The protein signals were quantified from the eletropherogram of the area under the chemiluminescent intensity peak obtained by Compass software.
Electrophysiological recordings. Whole  Ex vivo preparation. This technique is used to establish the relationship between storage volume and pressure. The bladders were extracted and the urethras ligated, close to the vesico-ureteric junctions. A catheter was placed in the urethral opening to record pressure and filling. Intravesical pressure was recorded in reference to atmospheric pressure. An amplifier was connected to a transducer that has been filled with water. A syringe filled with KRB solution was connected to a pump. The infusion rate of the KRB solution was 15-25 µl/min by automatic infusion and this was kept at 37 °C. The filling of the bladder with KRB solution was stopped when the pressure reaches 45-50 cmH 2 O. This is to avoid distending the bladder which can cause permanent tissue damage. Recordings obtained were analyzed by Clampfit (Molecular device) which had the baseline adjusted to examine the amplitude and frequency of pressures during filling (see Fig. 5). Statistical analyses. All data were expressed as means ± SEM. "n" denotes the number of animals used.

Solutions and chemicals. Whole-cell configuration was achieved in Ca
All statistical analyses were performed using Graphpad Prism. Student's paired or non-paired t test were used to compare groups of data and differences were considered to be significant at P < 0.05. Data analysis for the westerns was performed using Compass software (ProteinSimple, San Jose, CA, USA), and expressed as intensity area/ug of protein. Lane view images of the western blots were saved as JPEGs, opened with Adobe Photoshop, converted to TIFFs; and saved after adjusting the image resolution with the Auto Res function. www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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